Effect Of Berberine On The Expression Of Prostaglandin Receptors In High-fat Diet And Low-dose Streptozotocin-induced Diabetic Rats With Nephropathy

Objective：Through the study of berberine on the expression of prostaglandinreceptors in High-fat diet and Low-dose Streptozotocin-induced diabetic rats withnephropathy. In order to find out its meaningful prostaglandin receptor subtypes EP1,EP2, EP3and EP4in DN pathogenesis, And to explore the role of BBR on the possiblemechanism of DN rats.Methods: DN model in healthy male SD rats was induced with STZ onceintraperitoneal injection in a dose of35mg/kg after having high-fat diet for six weeks.After injected, all animals continued on the high-fat diet for72hours. The animals withfasting blood glucose(FBG) level of greater than or equal to11.1mmol/L and onlyuniformly diabetic rats were used in the study. Diabetic rats were randomly divided intosix groups: diabetic rats without any drug treatment(HFDC); diabetic rats treated withBBR at a dose of200,100and50mg/kg every day; diabetic rats treated with Enalaprilat a dose of1mg/kg; Xiaoke Wan at a dose of800mg/kg every day served as positivecontrol. Additionally, there were non-diabetic control (NC) rats that neither receivedSTZ nor the high-fat diet and high-fat diet control (HFC) rats. Except NC, HFC anddiabetic rats without any drug treatment were given an equal volume of vehicle(CMC-Na), while the other groups were given intragastrically drugs for8weeks. thedrug dose was adjusted according to the change of animal weight. All the rats wereallowed to continue to feed on their respective diets until the end of the study. Duringthe experiment, the rats’diet, drinking and urination situation were observed and body weight was measured every week. FBG was measured every2week on lateral tail veinblood samples. Eight weeks after the induction of diabetes, all rats were housedindividually within metabolic cages and urine was collected from the rats housed inmetabolic cages for24h. The overnight fasted rats were sacrificed. Blood samples wereobtained from femoral artery, centrifuged, and the supernatant was used formeasurement of glucose, lipid metabolic parameters and so on. kidney weight to bodyweight (KW/BW) ratio, total urine protein for24hours to urine creatinine（UP/C）ratio,urine microalbumin for24hours to urine creatinine（U.A/C）,blood urea nitrogen(BUN), serum creatinine (SCr). Separate kidney from the rats, HE staining andmasson’s trichrome staining were used to observe the pathological changes of kidney,and transmission electron microscopy was used to observe glomerular ultrastructure.The levels of interleukin-6(IL-6) and prostaglandin E2(PGE2) were measured byenzyme-linked immunosorbent assay(ELISA). Expression of E-prostanoidreceptors(EP1-EP4) were determined by Western blotting technique. Cyclic adenosinemonophosphate (cAMP) were detected by radioimmunoassay.Result：1. Effects of BBR treatment on body weight and fasting blood glucose of DN ratsThe high-fat diet increased BW compared with the NC diet throughout thetreatment period. Diabetic rats had lower body weights than HFC rats beginning atweek2. Diabetic rats had lower body weights than the NC rats beginning at week3.BBR treatment improved BW, but the differences between these groups did not reachstatistical significance during the entire study and remained lower than HFC rats. FBGwas significantly higher in diabetic than in nondiabetic rats. Treatment with BBR (100,200mg/kg) significantly decreased fasting blood glucose compared with untreateddiabetic groups from the end of the6th week to the end of8th week. 2. Effects of BBR on biochemical and renal functional parameters in DN ratsThe U.A/C, UP/C, BUN, SCr levels of HFDC group were significantly higher,This result indicates that our model was successful at inducing DN. When DN rats weretreated for8weeks, BBR (200mg/kg and100mg/kg) significantly decrease the levelsof U.A/C, UP/C, BUN, SCr. Data indicate that BBR treatment could effectivelyimprove renal function parameters and lipid metabolism in DN rats.3. Effect of BBR on renal histopathology of DN ratsCompared with NC group, the renal histology from untreated DN rats showedsigns of severe pathological changes such as mesangial matrix expansion andglomerular enlargement became more pronounced, and thickening of the glomerularbasement membrane was noticeable. In addition, deposition of ECM in the glomeruliwas aggravated, and there were abundant inflammatory cells in the tissues. Comparedwith the model group, administration of berberine was able to ameliorate mesangialmatrix expansion and to effectively ameliorate pathologic state.4. Effect of BBR on renal ultrastructureThe electron micrograph shows a normal ultrastructure of the glomeruli in NC. Inthe DN model rats, the glomeruli appears larger and dilated, the GBM was wrinkled andthickened, local fusion of the foot processes of the podocytes and podocytes loss wereobserved compared with NC group. Oral administration of BBR (200mg/kg and100mg/kg) could normalize the above mentioned alterations to some extent.5. Effect of BBR on the level of IL-6and PGE2The enzyme-linked immunosorbent assay showed that the level of IL-6wassignificantly increased in the DN model group compared with the normal group.Treatment of BBR (200mg·kg and100mg·kg) significantly reduced the level of IL-6 compared with the DN model group. The production of PGE2was apparently increasedin the DN model group compared with the normal group. Treatment of BBR (200mg·kg and100mg·kg) markedly depress the production of PGE2compared with theDN model group.6. Effects of BBR on the production of EP1、EP2、EP3and EP4in renal cortexBy Western blot assay, EP1,EP2,EP3and EP4which are expressed in kidney, wereanalyzed by western blotting. The protein expression levels of EP1and EP3wereenhanced apparently, while the expression of EP4was reduced sharply in renal cortex ofDN model group compared to controls. Treatment with BBR (200mg/kg/d,100mg/kg/d)not only down-regulate the protein expression of EP1and EP3, but also up-regulationprotein expression of EP4level was slightly increased after BBR treatment, but thisdifference did not reach statistical significance.7. Effectsof BBR on the level of cAMPBy radioimmunoassay, the level of cAMP was found to be transparently decreasedin the DN model group compared with the normal group. The BBR treatment groups(200mg·kg and100mg·kg) had visibly increased levels of cAMP compared with theDN model group.Conclution:1. The characteristics of therapeutic effects of BBR on DN rats mainly shows in thatBBR could improved renal function parameters, and improved fasting blood glucosesignificantly after a long-term treatment, but has no obvious effect on the weight ofDN rats.2. BBR administration can significantly reduce the levels of IL-6and PGE2in DN rats. 3. The expression of EPs protein from the renal cortical of DN rats and the level ofcAMP were both abnormal, BBR administration can regulate the expression of EPs, italso raised the level of cAMP. PGE2-EP-cAMP have abnormal changes in DN rats, sowe speculate that BBR may play a key role in the prevention and treatment of DN byadjusting the PGE2-EP-cAMP normal transduction.